I have performed the TNF alpha ELIspot for around 15 experiments. It is a commercial kit based protocol. I am facing this problem of no spot development for the last two assays. The same samples have been used in my previous assays which have worked out. The cell density, incubation time and stimulant concentrations have been previously optimized.
The appearance of a blue background and no spot development in a Human TNF alpha ELISpot assay could be due to several reasons:
1. Inadequate cell stimulation: Ensure that the cells used in the assay are appropriately stimulated with the appropriate activators or antigens to induce TNF alpha production.
2. Incorrect cell density: Make sure to use the correct cell density when setting up the ELISpot assay. Too few or too many cells can affect spot development.
3. Improper plate coating: Check that the ELISpot plate is coated with the capture antibody for TNF alpha properly. Insufficient coating or incorrect antibody concentration can lead to poor spot development.
4. Detection antibody issues: Confirm that the detection antibody used is specific to human TNF alpha and at the correct concentration.
5. Substrate development: Ensure that the substrate used for the ELISpot assay is fresh and properly prepared. Aging or improper preparation of the substrate can lead to a blue background.
6. Overdevelopment: If the assay is left to develop for too long, it can lead to high background staining.
7. Contamination: Ensure that all reagents and equipment used in the assay are free from contamination.
To troubleshoot the issue, it is recommended to carefully review the assay protocol, recheck all reagents, and consider performing control experiments to identify the source of the problem. If the issue persists, consulting with experienced researchers or technical support from the ELISpot kit manufacturer might be helpful.
Sometimes, the antigen-specific T cells of interest have a very low magnitude that impede their detection or activity (cytokine secretion) even upon stimulation.
In the past, I had the same issue to detect IFNg-secreting T cells derived from PBMCs of macaques infected with lymphocryptovirus. To overcome that problem, I had to cultivate PBMCs with a cytokine cocktail for 14 days before adding the cells into the Elispot plates. More info about that strategy can be found in the following article:
https://pubmed.ncbi.nlm.nih.gov/24089556/
Therapeutic vaccination against the rhesus lymphocryptovirus EBNA-1 homologue, rhEBNA-1, elicits T cell responses to novel epitopes in rhesus macaques
Eduardo L V Silveira et al. J Virol. 2013
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